Inokuchi Yuta, Imai Shunsuke, Nakajima Yoshimi, Shimazawa Masamitsu, Aihara Makoto, Araie Makoto, Hara Hideaki
Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, Gifu, Japan.
J Pharmacol Exp Ther. 2009 May;329(2):687-98. doi: 10.1124/jpet.108.148676. Epub 2009 Feb 6.
Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger, is used for the treatment of acute cerebral infarction. In this study, we investigated whether edaravone is neuroprotective against retinal damage. In vitro, we used a radical-scavenging capacity assay using reactive oxygen species-sensitive probes to investigate the effects of edaravone on H(2)O(2), superoxide anion (O(2)*), and hydroxyl radical (*OH) production in a rat retinal ganglion cell line (RGC-5). The effect of edaravone on oxygen-glucose deprivation (OGD)-induced RGC-5 damage was evaluated using a 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt assay of cell viability. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) significantly decreased radical generation and reduced the cell death induced by OGD stress. In vivo, retinal damage was induced by intravitreous injection of N-methyl-D-aspartate (NMDA; 5 nmol) and was evaluated by examining ganglion cell layer cell loss, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, and the expressions of two oxidant-stress markers [4-hydroxy-2-nonenal (4-HNE) and 8-hydroxy-2-deoxyguanosine (8-OHdG)]. In addition, activations of mitogen-activated protein kinases (MAPKs) [extracellular signal-regulated protein kinases (ERK), c-Jun NH(2)-terminal kinases (JNK), and p38 MAPK], as downstream signal pathways after NMDA receptor activation, were measured using immunoblotting and immunostaining. Edaravone at 5 and 50 nmol intravitreous injection or at 1 and 3 mg/kg i.v. significantly protected against NMDA-induced retinal cell death. At 50 nmol intravitreous injection, it 1) decreased the retinal expressions of TUNEL-positive cells, 4-HNE, and 8-OHdG and 2) reduced the retinal expressions of NMDA-induced phosphorylated JNK and phosphorylated p38 but not that of phosphorylated ERK. These findings suggest that oxidative stress plays a pivotal role in retinal damage and that edaravone may be a candidate for the effective treatment of retinal diseases.
依达拉奉（3 - 甲基 - 1 - 苯基 - 2 - 吡唑啉 - 5 - 酮）是一种自由基清除剂，用于治疗急性脑梗死。在本研究中，我们调查了依达拉奉对视网膜损伤是否具有神经保护作用。在体外，我们使用活性氧敏感探针进行自由基清除能力测定，以研究依达拉奉对大鼠视网膜神经节细胞系（RGC - 5）中过氧化氢（H₂O₂）、超氧阴离子（O₂⁻）和羟自由基（·OH）生成的影响。使用2 - （2 - 甲氧基 - 4 - 硝基苯基） - 3 - （4 - 硝基苯基） - 5 - （2,4 - 二磺酸苯基） - 2H - 四唑单钠盐细胞活力测定法评估依达拉奉对氧 - 葡萄糖剥夺（OGD）诱导的RGC - 5损伤的作用。依达拉奉（3 - 甲基 - 1 - 苯基 - 2 - 吡唑啉 - 5 - 酮）显著减少自由基生成，并减少OGD应激诱导的细胞死亡。在体内，通过玻璃体内注射N - 甲基 - D - 天冬氨酸（NMDA；5 nmol）诱导视网膜损伤，并通过检查神经节细胞层细胞丢失、末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）染色以及两种氧化应激标志物[4 - 羟基 - 2 - 壬烯醛（4 - HNE）和8 - 羟基 - 2 - 脱氧鸟苷（8 - OHdG）]的表达来评估。此外，使用免疫印迹和免疫染色测量丝裂原活化蛋白激酶（MAPK）[细胞外信号调节蛋白激酶（ERK）、c - Jun氨基末端激酶（JNK）和p38 MAPK]作为NMDA受体激活后的下游信号通路的激活情况。玻璃体内注射5和50 nmol或静脉注射1和3 mg/kg的依达拉奉可显著保护视网膜细胞免受NMDA诱导的死亡。在玻璃体内注射50 nmol时，它1）降低了视网膜中TUNEL阳性细胞、4 - HNE和8 - OHdG的表达，2）降低了视网膜中NMDA诱导的磷酸化JNK和磷酸化p38的表达，但未降低磷酸化ERK的表达。这些发现表明氧化应激在视网膜损伤中起关键作用，并且依达拉奉可能是有效治疗视网膜疾病的候选药物。
